Epithelial to mesenchymal Hedgehog (Hh) signaling is critical for intestinal development and homeostasis. Hedgehog paralogs Sonic (Shh) and Indian (Ihh) are expressed at low levels in intestinal epithelium into adulthood, although their role in intestinal homeostasis is poorly understood. Reporter animals that express lacZ in cells with active Hh signaling reveal that myeloid cells and myofibroblasts are among the cellular targets of the Hh signal in the lamina propria. Indeed, mouse models of chronic Hh inhibition or conditional knockout of Ihh in the adult intestine exhibit blunted villi, crypt hyperplasia, mislocalization of myofibroblasts and spontaneous inflammation. These data suggest that Hh ligands promote anti-inflammatory responses in lamina propria mononuclear cells. This proposal aims to further delineate the role of Hh in adult intestinal homeostasis by elucidating the acute effects of Hh signal transduction on inflammatory signaling in vitro and in vivo. We hypothesize that Hedgehog signals secreted by intestinal epithelial cells influence lamina propria myeloid cells and myofibroblasts, leading to tolerogenic conditioning. Conversely, decreased hedgehog signaling activates mucosal associated myeloid cells and myofibroblasts, facilitating inflammation. This hypothesis will be tested by examining the effect of acutely decreased or increased Hh exposure on the phenotype of small intestinal lamina propria cells. First, mouse models in which conditional intestine-specific Hh inhibition or activation can be induced will be employed to examine the effect of altered Hh levels on the phenotype of myeloid cells and myofibroblasts in the lamina propria. We will test whether the timing or the level of Hh signaling has the greater impact on inflammatory phenotype. Second, we will determine whether Hh ligand is sufficient to promote tolerance in CD11b+ myeloid cells, including dendritic cells and macrophages, in vitro; quantitative real-time RT-PCR, ELISA assays FACS analysis and T-cell activation assays will be performed to determine the cytokine secretion, co-receptor expression and overall phenotype of cells isolated from low, high and normal Hh signaling environments. As a corollary to this question, we will examine whether bone marrow-derived monocytes can be tolerogenically conditioned with Hh ligands. Finally, myofibroblasts from low, high and normal Hh environments will be analyzed for phenotypic changes, particularly in their non-professional immune cell functions. This work will vastly improve our understanding of the complex interaction of lamina propria myeloid cells and myofibroblasts with epithelial derived Hh signals. The work will examine these Hh target cells with respect to cytokine output and inflammatory phenotype as well as with respect to their ability to modulate the adaptive immune system, a system that is implicated in a myriad of autoimmune and inflammatory diseases. PUBLIC HEALTH RELEVANCE: Hedgehog (Hh) signals are critical for multiple events during intestinal development and are downregulated, but still present in adult life in some organs, including the GI tract. In the adult intestine, myofibroblasts and myeloid cells are direct cellular targets of Hh signaling. These cells can modulate the inflammatory milieu and communicate with T-cells and are thus important integrators of inflammatory signaling. Indeed, preliminary studies indicate that Hh signals promote a tolerogenic phenotype in these cells. The goal of this work is to determine how Hh affects functional inflammatory signaling in myeloid cells and myofibroblasts. These studies will increase our understanding of how Hh influences intestinal immune responses, information that may be highly relevant to inflammatory bowel disease.